Mutagenesis is an important tool for studying evolution on a laboratory time scale and for developing new biomolecules through directed evolution. Current mutagenesis strategies in living systems are largely global, meaning that an entire organism is mutated rather than just the gene of interest. The resulting off-target mutations can be cytotoxic, which requires a careful balance between maximizing the extent of mutagenesis while minimizing cell death. This balance puts a constraint on library size. Furthermore, off-target mutations can allow an organism to circumvent a selection pressure in an undesired way in the context of directed evolution. To overcome these challenges, we have devised a system that targets mutations only to a particular gene of interest. Specifically, using a DNA damaging enzyme fused to an RNA polymerase, we can target mutations to a specific region of DNA rather than the entire genome. This construct should allow for continuous evolution experiments to be carried out on specific genes in a broad array of organisms from <i>E. coli</i> to human cells.<br/>

Targeting mutations to specific genes in living systems

255th National Meeting (2018)

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